cGMP, an intracellular secondary messenger, plays an important role in regulating various cellular activities. It is converted from GTP by soluble guanylate cyclase (sGC) and broken down by phosphodiesterases (PDEs). Thus, elevation of the cGMP levels can be achieved by increasing the activity of sGC or reducing the activity of PDEs.
Platelet aggregation contributes to the pathogenesis of various cardiovascular diseases, e.g., atherosclerosis, myocardial infarction, unstable angina pectoris, thrombosis, and hypertension. As low intracellular levels of cGMP cause enhanced platelet aggregation, increasing cGMP levels in platelets provides a way of treating these diseases. Intracellular cGMP levels are also known to influence other physiological functions, e.g., penile erection.
Compounds that boost the intracellular cGMP levels, either by activating sGC or by inhibiting PDEs, have clinical implications for disorders related to low intracellular cGMP levels. Certain pyrazolyl compounds have been found to activate sGC and are potential cardiovascular drugs.
An aspect of the present invention relates to novel fused pyrazolyl compounds of formula (I): 
wherein each of Ar1, Ar2, and Ar3, independently, is phenyl, thienyl, pyrrolyl, or furyl, optionally substituted with halo, alkyl, carboxyl, alkoxycarbonyl, thiocarbonyl, aminocarbonyl, hydroxyalkyl, alkoxyalkyl, aminoalkyl, or thioalkyl; each of X and Y, independently, is O, S, or NH; m is 1, 2, or 3; and n is 0, 1, 2, 3, or 4. The term xe2x80x9calkyl,xe2x80x9d the prefix xe2x80x9calkxe2x80x9d (as in alkoxyalkyl), or the suffix xe2x80x9c-alkylxe2x80x9d (as in hydroxyalkyl) refers to C1-6.
Referring to formula (I), a subset of the compounds of this invention are featured by that X is O, Y is O, and m is 1. In these compounds, Ar2 is preferably phenyl or furyl, or Ar3 is thienyl or phenyl. Another subset of the compounds of this invention are featured by that Ar2 is phenyl or furyl. In these compounds, Ar1 is phenyl, or Ar3 is thienyl or phenyl. Still another subset of the compounds of this invention are featured by that Ar3 is thienyl or phenyl. In these compounds, Ar1 is phenyl and Ar2 is furyl; or Ar2 is phenyl.
Four exemplary compounds of this invention are 1-benzyl-3-(5xe2x80x2-methoxycarbonyl-2xe2x80x2-furyl)-5,6-methylenedioxoindazole, 1-benzyl-3-(5xe2x80x2-hydroxycarbonyl-2xe2x80x2-furyl)-5,6-methylenedioxoindazole, 1-benzyl-3-(5xe2x80x2-methoxymethyl-2xe2x80x2-furyl)-5,6-methylenedioxoindazole, and 1-benzyl-3-(5xe2x80x2-hydroxymethyl-2xe2x80x2-furyl)-5,6-methylenedioxoindazole. The structure of 1-benzyl-3-(5xe2x80x2-hydroxycarbonyl-2xe2x80x2-furyl)-5,6-methylenedioxoindazole is shown below, with the atoms in the aryl rings numbered: 
The fused pyrazolyl compounds described above include their salts, if applicable. Such a salt, for example, can be formed between a positively charged substituent, e.g., amino, and an anion. Suitable anions include, but are not limited to, chloride, bromide, iodide. sulfate, nitrate, phosphate, or acetate. Likewise, a negatively charged substituent (e.g., carboxylate) can form a salt with a cation. Suitable cations include, but are not limited to, sodium ion, potassium ion, magnesium ion, calcium ion, and an ammonium cation such as tetramethylammonium ion. Two examples of salts of this invention are the hydrochloride salt of 1-benzyl-3-(5xe2x80x2-aminomethyl-2xe2x80x2-furyl)-5,6-methylenedioxoindazole and the sodium salt of 1-benzyl-3-(5xe2x80x2-carboxyl-2xe2x80x2-furyl)-5,6-methylenedioxo indazole.
Compounds of this invention can activate sGC or inhibiting PDEs.
Thus, another aspect of the present invention relates to a pharmaceutical composition containing an effective amount of a compound of formula (I) and a pharmaceutically acceptable carrier for treating diseases related to low activity of sGC, high activity of PDE, or platelet aggregation.
Details of several embodiments of the invention are set forth in the description below. Other features, objects, and advantages of the invention will be apparent from the description, and also from the claims.
A fused pyrazolyl compound of this invention can be synthesized by the following method. React an alkylenedioxoarylacyl chloride with an aryl compound to produce an alkylenedioxoaryl aryl ketone. The ketone is then reacted with a hydrazine to produce a hydrazone, which is subsequently converted to an intermediate in the presence of a first catalyst Pb(OAc)4. Without being purified, the intermediate is further converted to a fused pyrazolyl compound in the presence of a second catalyst BF3.Et2O. Desired functional groups, e.g., hydroxy carbonyl or hydroxyalkyl, can be introduced into the fused pyrazolyl compound thus obtained by further modifications.
Shown below is a scheme which depicts the synthesis of four fused pyrazolyl compounds 1, 2, 3, and 4 of this invention: 
Details of synthesis of compounds 1, 2, 3, and 4 are described in Examples 1, 2, 3, and 4, respectively.
Compounds of this invention can be used to increase the intracellular levels of cGMP by activating sGC or inhibiting PDEs. Thus, another aspect of this invention relates to a pharmaceutical composition which contains an effective amount of at least a fused pyrazolyl compound of formula (I) (or its salt) and a pharmaceutically acceptable carrier for treating diseases associated with low intracellular cGMP levels, e.g., impotence or platelet aggregation-related disorders. xe2x80x9cAn effective amountxe2x80x9d refers to the amount of the compound which is required to confer a therapeutic effect on the treated subject. The interrelationship of dosages for animals and humans (based on milligrams per meter squared of body surface) is described in Freireich et al., Cancer Chemother. Rep., 1966, 50, 219. Body surface area may be approximately determined from height and weight of the patient. See, e.g., Scientific Tables, Geigy Pharmaceuticals, Ardley, N.Y., 1970, 537. Effective doses will also vary, as recognized by those skilled in the art, depending on route of administration, excipient usage, and the possibility of co-usage with other therapeutic treatments including use of other anti-platelet aggregation agents. Examples of the carriers include colloidal silicon dioxide, magnesium stearate, cellulose, sodium lauryl sulfate, and DandC Yellow #10.
The pharmaceutical composition may be administered via a parenteral route, e.g., topically, subcutaneously, intraperitoneally, intramuscularly, and intravenously. Examples of parenteral dosage forms include aqueous solutions of the active compound, in an isotonic saline, 5% glucose, or any other well-known pharmaceutically acceptable carrier. Solubilizing agents, such as cyclodextrins, or other solubilizing agents well known to those familiar with the art, can also be included in the pharmaceutical composition.
A fused pyrazolyl compound of this invention can be formulated into dosage forms for other routes of administration (e.g., orally, mucosally, or percutaneously) utilizing well known methods. The pharmaceutical composition can be formulated, for example, in dosage forms for oral administration in a capsule, a gel seal, or a tablet. Capsules may comprise any well known pharmaceutically acceptable material such as gelatin or cellulose derivatives. Tablets may be formulated in accordance with the conventional procedure by compressing mixtures of the active compounds, a solid carrier, and a lubricant. Examples of solid carriers include starch and sugar bentonite. The compound can also be administered in a form of a hard shell tablet or capsule containing, for example, lactose or mannitol as a binder, a conventional filler, and a tableting agent.
Also within the scope of this invention is the use of a fused pyrazolyl compound of formula (I) for the manufacture of a medicament for the uses described above.
The compounds of this invention can be preliminarily screened for their efficacy in treating the above-described diseases by one or more of the following in vitro assays:
The efficacy of a compound in activating sGC can be evaluated in vitro by the following assay. Washed platelets are suspended in a buffer and disrupted by sonication. The lysate is then centrifuged to obtain a supernatant which is used as the source of sGC. An aliquot of the supernatant and the compound to be tested are added into a buffer containing GTP, a substrate for sGC. The activity of sGC can be determined by the method described in Gerzer et al., J. Pharmacol. Exp. Ther. 1983, 226:180.
The efficacy of a compound in inhibiting PDEs can be evaluated in vitro by the following assay. Washed platelets are suspended in a Tris-HCl buffer and disrupted by sonication. The lysate is centrifuged to obtain a supernatant which contains PDEs. An aliquot of the supernatant is taken to prepare a PDE-containing solution. The compound to be tested and cGMP (a substrate for PDE) are added to the solution. Ophiophagus hannah snake venom is subsequently added to remove the phosphate in 5xe2x80x2-GMP (converted from cGMP by PDEs) to form uncharged guanosine. An ion-exchange resin is used to remove the remaining cGMP. The cGMP-free solution is then centrifuged, and an aliquot of the supernatant is taken for quantification of the uncharged guanosine in a liquid scintillation counter. Activity of PDEs is evaluated based on the amount of the uncharged guanosine.
In vitro assays can be used to evaluate the efficacy of a fused pyrazolyl compound of this invention in inhibiting platelet aggregation; which is attributable to low intracellular cGMP levels. For example, the compound is incubated in a platelet suspension containing a platelet aggregation factor, and the degree of aggregation is measured turbidimetrically with a dual-channel lumiaggregometer and converted into a percentage value by the method described in Teng et al.; Biochem. Biophys Acta. 1987, 924:375-382.
In vivo screening can be performed by following procedures well known in the art.
Without further elaboration, it is believed that one skilled in the art can, based on the description herein, utilize the present invention to its fullest extent. All publications recited herein are hereby incorporated by reference in their entirety. The following specific examples, which describe synthesis and biological testing of various compounds of the present invention, are therefore, to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever.